1. Field of the Invention
This invention relates generally to a method for gene-selection, and more specifically to a method of preselecting a specific recombinant vector containing a nucleic acid sequence of interest from a multitude of different recombinants, and direct introduction of the preselected recombinant vector into a competent host cell.
2. Background and Related Art
The industrial applications of genetic engineering are becoming evident in the production of pharmaceuticals, of foods having improved properties, and of chemical products (including enzymes) to facilitate manufacturing processes. The process of genetic engineering may begin by cloning a gene of interest which encodes a protein with the desired properties for the particular industrial application. Typically, cloning a gene is done by either breaking up a genome into manageable sized fragments, or generating cDNA fragments from isolated m-RNA, and then cloning those genomic or cDNA fragments into a vector and transforming the resultant recombinant vectors into a competent host cell. Commonly used methods for screening transformants, to identify a transformant that contains the recombinant vector with the desired nucleic acid sequence or gene of interest, include a colony-filter lift process where either:
(a) the colonies are lysed on the filter and the filter is screened for the expression of the gene product of interest using monoclonal or polyclonal antisera followed by treatment with a conjugate and subsequent substrate development; or PA1 (b) the colonies are lysed onto the filter, and the colony DNA remaining on the filter is denatured and then hybridized to an oligonucleotide probe having incorporated a nonisotopic or isotopic label, wherein the probe is comprised of a nucleic acid sequence complementary to the nucleic acid sequence or gene of interest. Depending on the label incorporated into the probe, the screening process is completed by subjecting the hybridized filter to either substrate development or autoradiography. A modification of this latter technique has been described in U.S. Pat. No. 4,865,967.
However, the cloning of a specific nucleic acid sequence or gene is difficult, tedious, and time-consuming because of the ratio of the occurrence of that sequence or gene relative to the size of the genome or cDNA library to be screened. Thus, in using a colony-filter lift process, usually multiple filters are screened in attempts to identify a single colony containing a recombinant vector with the sequence or gene of interest. Additionally, if screening is done by a method requiring expression of a desired gene product, screening is complicated by the requirements that the gene be expressed in that vector and by the transformed microorganism in a form recognizable by the screening antiserum, and that the expressed gene product is not lethal for the host cell.
A method of physically isolating a recombinant plasmid of interest from a mixture of plasmids has been described (Rigas et al, 1986, Proc. Natl. Acad. Sci. USA, 83:9591-9595). In this method, Rec-A was used to form stable complexes between a single-stranded biotinylated probe and the double-stranded DNA molecules sharing sequence homology. Avidin is added to the reaction solution to bind to the probe, and the reaction mixture is chromatographed over cupric iminodiacetic acid agarose beads with a recovery of desired plasmid ranging from 10-20%. A method of preselecting, wherein the loss of preselected nucleic acid is minimized, is desirable.